Laser processing and manufacturing of micro‐ and nanoscale biosystems
F. Sima1, O. Gallet2, K. Anselme3, I. N. Mihailescu4, K. Sugioka5
1Center for Advanced Laser Technologies (CETAL), National Institute for Laser, Plasma and Radiation Physics, Romania2Equipe de Recherche sur les Relations Matrice Extracellulaire‐Cellules, ERRMECe, University of Cergy‐Pontoise, France
3Institut de Science des Matériaux de Mulhouse (IS2M), CNRS UMR7361, Mulhouse, France4Lasers Department, National Institute for Lasers, Plasma and Radiation Physics (INFLPR), Magurele, Romania
5RIKEN Center for Advanced Photonics, Wako, Saitama 351‐0198, [email protected]
June 12‐14, 2019, Bucharest, RomaniaAn event of the Romanian Presidency of the Council of the European Union
E. Axente1, F. Jipa1, S. Iosub1, C. Pana1, C. Butnaru1
Miniaturized platforms for bio‐related applicationsMotivation:1. boosting sensing capabilities by bio‐mimicking and reducing sizes in 2D & 3D configurations;2. replacing conventional methods by innovative technologies to reduce cost and propose new characteristics.
http://www.dailycal.org
Mimic organs
2D
3D
Trends Cell Biol. 2011, 21(12): 745
Biomimetic microsystems –single biochip ‐microfluidic circulatory 3D system drugabsorption –evaluate drug efficacy and toxicity.
Devices that require robustness, biocompatibility and functionality(if possible, multiple use) – Lab on a Chip (LoC), Point of Care (PoC)
Biopsy Sample
Testµ‐chamber
www.beyondpositive.orghttp://web.anl.gov
HIV detectionCancer identification
http://kyocera‐md.com/technology/surface/
Localized implant coatings
Conventional techniques to deposit thin organic coatings
Spin coating
Water
Langmuir–BlodgettLbL
polar head(hydrophilic)
non-polar head
Difficult to obtain multistructure-layers
Material consuming techniques;Substrate limited;
Conventional biochip 3D platforms: current approach and limitations of 3D cell culture to organs‐on‐chips
Fabrication methods for microfluidic chips.Pros: Most chips are made out of PDMS because it is easy to use and has high optical clarity, gas permeability and biocompatibility.
Cons: PDMS can adsorb small organic compounds, including many drugs, and its high gas permeability can hinder some applications.
PDMS - poly-dimethylsiloxane
Nature Biotechnology, 32, 760 (2014)
**Thermalization time is of 1-100 ps
Heat Affected Zone (HAZ)
hυ≥Eg
Minor Affected Zone
Melting(zone extension)
Material surface Material volume
Explosion
1. Short pulses (inferior to few ps)
2. Long pulses(superior to few ps)
Non‐thermal process
Thermal diffusion
Multi‐photon Nonlinear absorption
Linear absorption
3D modificationwith high resolution
High density photons
High energy photons
hυ<Eg
Unconventional: laser pulses
*Ultrashort = femtosecond (fs) and few picosecond (ps)
**energy deposition timescale shorter than electron‐phonon coupling processes
ultrashort*
non-continuous wave with power concentrated in a pulse of some duration
Outline1. Biomimetic 2D coatings grown by pulsed lasers – long energetic pulses
2. Biomimetic 3D environments fabricated by pulsed lasers – ultrashort pulses
Biomimetism related to composition – structure ‐ properties
Biomimetism related to architectural and fluid flow aspects
Part I
Biomimetic 2D coatings grown by pulsed laser depositions techniques
8
E=hν=hc/λ
Laser‐matter interaction – long pulses
Crater
Photons
Vapors
Charged particles(e-, ions, plasma)
Thermal wave
Shock wave
Matter(solid, liquid, gas)
Laser beamApplications
Generation of nanoparticles
Thin nanofilms and coatings
Surface modification
Micro- and Nanopatterning
Fundamental aspects
Laser deposition techniques of thin coatingsPulsed Laser Deposition (PLD)
Inorganic thin filmsMatrix Assisted Pulsed Laser Evaporation (MAPLE)
Organic thin films
active material (solute) is dissolved in a solvent (matrix) the liquid mixture is transformed in solid by freezing (in LN); target kept at low temperature with a cooler during deposition (100 -200 K).
Main difference between PLD and MAPLE:
target and implicitly the mechanism of laser - material interaction
Applied Surface Science 418, 580, 2017
Liquid N2
Substrate
Volatile solvent molecules are pumped away
Cooler
Frozen target
Heater
FN
x
y
MAPLE of fibronectin (FN): “dry” process?
Solution FN (0.25-2mg/ml)in water buffer (TRIS and NaCl) at pH 7.4
Laser:λ=248 nm, 25 ns;15 HzE=140 mJ;Spot A: 18 mm2;N. pulses: 20000
Other parameters:Dist. T‐S = 30 mm;Temperature 30 oC;Pressure: 10‐1 mbarrSubstrates: Si, Ti, HA* coated Ti;
Acta biomaterialia 7 (10), 3780, 2011
Titanium
Osteoblast progenitor cells
nanostructured HA* layersprotein coating
Titanium
protein coating
Osteoblast progenitor cells
HA* ‐ hydroxyapatite, main inorganic component of the bone;Ca2+ ions of HA are binding sites for FN molecules
Fibronectin coatings on Ti and HA coated Ti: why using HA as interlayer?
Ca10(PO4)6(OH)2 ‐Applied Physics A 105 (3), 611, 2011
In vitro evaluation: osteoprogenitor cells
For all coatings, the number of cells is more orless constant in the first few days, until day 7 afterwhich the number of cells increases. Between day14 and 21 a relative decline in proliferation isobserved which in the case of osteoprogenitorcells is quite systematic behavior.
FN induces a higher degree of differentiation atday 21 in comparison with the cells grown onHA or HA-BSA structures. Less than 7 µg of FNper cm2 deposited by MAPLE on HA coatingsimprove cell differentiation compared to HAcoatings alone.
ACS applied materials & interfaces 7 (1), 911, 2015 BSA* ‐ Bovine Serum Albumin – inert protein
*
*
Gradient M1‐M2 by Combinatorial MAPLE
d T-S
Ts
Solution M1
Solution M2
M1
Applied Physics Letters 101 (23), 233705, 2012
M2
Innovative design: ring‐like concentric holder
substrate area
Protein blends by C-MAPLE
Applied Surface Science 306, 75, 2014
Surface activationDrug delivery systems
Conclusions:
1. The safe laser transfer of a large molecular mass protein – intactand functional;
2. Introducing a hybrid biomimetic inorganic –organic system;
3. Development of a combinatorial laser approach (C‐MAPLE) forgrowth of gradient organic thin films with variable composition orfor active material release from polymeric matrix.
Part II
Biomimetic 3D environments fabricated by pulsed laser technologies
Ultrashort laser pulses – high enough peak intensities (around 1013 W/cm2)
3D micro and nano processing based on multiphoton absorption using ultrashort lasers
Laser direct write Chemical etchingFabricated 3‐D structure
Substrate
Photo curable resin
Subtractive manufacturing Additive manufacturing
Femtosecond laser assisted etching (FLAE) of glass Two‐photon polymerization (TPP)Hybrid subtractive and additive manufacturing will further enhance performance of femtosecond 3D microprocessing.
Initiation: 𝐼 2ℎ𝜐 → 2𝑅 • =>𝑅 • 𝑀 → 𝑅𝑀 •,
Propagation : 𝑅 𝑀𝑛 • 𝑀 → 𝑅 𝑀𝑛 •,
Termination: 𝑀 • •𝑀 → 𝑀 𝑀
nEph > Eg
Hybrid FLAE‐TPP to integrate polymeric patterns inside glass:photosensitive glass ‐ negative photoresist
The hybrid process (FLAE‐TPP) allows lowering the size limit inside channels to smaller details, improve thestructure stability in the same time as it offers the required robustness for assembling a concrete LoC device.
sub‐diffraction‐limitspatial resolution ispossible in a thresholdsystem in whichmaterial responds tolight excitation with apronounced thresholdbehavior.
Micromachines 8 (2), 40, 2017; Nanophotonics 7 (3), 613, 2018
Scale up scale down aspects
Microfluidic gradient generatorsfor chemotaxis in research laboratory
Geometric design of a gradient forming region, concentration of a source and a sink andrelative degree of molecular influx and outflux determine the gradient profile.Understanding cellular behavior such as orientation and migration as a whole population.
Integr. Biol., 2010, 2, 584–603
Aim: reducing size in glass, fabrication of very narrow channelsbioplatform for cell chemotaxis
http://www.chemotaxis.usn.hu
flow‐based gradient generators
diffusion‐based gradient generators
Cell response to external stimuli(organism)
Migration Infection site
design‐site.net
Adherent cells
Use of narrow channels – cancer cell invasiveness and migration study
Early stages of metastasis formation and cancer cell invasion. During migration and invasion, cells must undergo largemorphological changes in order to cross the basement membrane and move through connective tissue.
Understanding cancer cell invasion and migration in 3D closed µ‐environments
https://www.pinterest.com/cancerhallmarks/cancer‐metastasis/
Science, 2016; 352(6283):353
Cell migration incurs substantial physical stress on the nuclear envelope and its content and requires damage repair for cell survival.
Size reduction challenging in glass µ‐channels – innovative scaffoldfor single cell manipulation and analysis
µ‐ and subµ Cell nucleus
Cell of 20 µm diameter
improvement of TPP resolution
ACS Applied BioMaterials, 2018
Size reduction challenging in glass µ‐channels – innovative scaffoldfor single cell manipulation and analysis
3D confined spaces fabricated using TPP: laser tailoring downsizing ACS Applied BioMaterials, 2018
Size reduction challenging in glass µ‐channels for single cell manipulation and analysis – design proposal
Hierarchical biochip design and concept
Substractive-additive processesobservation area and scheme for in vitro chemoattractant gradient generation for experimental testing of cancer cell migration potential in confined spaces.
Size reduction challenging in glass µ‐channels for single cell manipulation and analysis
For a better observation and channel size evaluation inside glass, the microfluidic system was filled with SU‐8 photoresist to compensate refractive index mismatch
Gradient formation: fluorescein test without and with scaffold
Movie
Funnel effect Funnel effect + filter
Scale up ‐ scale down
1. Measure
2. Analyze
3. Improve
4. Control
1. Measure
2. Analyze
3. Improve
4. Control
5. Slow release
Fluorescein has an absorption maximum at 494 nm and emission maximum of 512 nm (in water)
Time lapse imagining of cancer cells (PC3): area of observation
1. Cells loaded and grown inside channels for few days to increase their density;2. Cells were starved over night (by using cellular media without FBS);3. EGF* was added next day and orientation and migration monitored for 3 h.
*epidermal growth factor
µ‐channel array scaffold (area of observation)
Time lapse imagining of cancer cells (PC3) responding to chemo‐gradient
Time lapse imagining of cancer cells (PC3) responding to chemo‐gradient
PC3 cell migration through 3D sub‐micron‐scale confined spaces: a) red arrow indicates the exit of the 3rd channel from the left from which the first cell will appear; b) the first PC3 cell isdisintegrating after migration; c) first PC3 cell (red arrow) is reintegrating after migration while a second cell (yellow arrow) appears at the exit of the 4th channel; d) first PC3 cell (redarrow) is still reintegrating after migration while the second cell (yellow arrow) is spreading and disintegrating; e) first PC3 cell (red arrow) is reducing motility, second cell (yellow arrow)is reintegrating after migration while the third cell (blue arrow) appears at the exit of the 6th channel; f) the second PC3 cell (yellow arrow) fusing with third cell (blue arrow).
Conclusions
1. Ultrashort laser hybrid method allows both photosensitiveglass 3D processing and TPP additive polymer integrationinside glass microchannels for true 3D “ship‐in‐a‐bottle”biochips.
2. Capability of 3D micro‐ and nanofabrication of fluidicsystems by combining the advantages of individual specificcharacteristics and compensating the drawbacks (e.g.structure stability and functional device assembling).
3. Polymeric micro‐ and nanostructure pattern integrationinside microfluidic systems covers the scale‐down ‐ scale‐upaspects of a multi‐functional microfluidic device, manipulationon both 2D and 3D environments and optical visualization,increase in sensitivity and eventually in the performance ofassembled devices.
Thank you for your attention!